© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 4448-4456 doi:10.1242/dev.113654
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Downregulation of miR122 by grainyhead-like 2 restricts the
hepatocytic differentiation potential of adult liver progenitor cells
Naoki Tanimizu*, Seiji Kobayashi, Norihisa Ichinohe and Toshihiro Mitaka
Late fetal and adult livers are reported to contain bipotential liver stem/
progenitor cells (LPCs), which share surface markers, including
epithelial cell adhesion molecule (EpCAM), with cholangiocytes and
differentiate into both hepatocytes and cholangiocytes. However,
recent results do not necessarily support the idea that LPCs
contribute significantly to cellular turnover and regeneration by
supplying new hepatocytes. Here, we examined the colony-forming
capability of EpCAM+ cells isolated from mouse livers between E17
and 11 weeks of age. We found that the number of bipotential
colonies was greatly reduced between 1 and 6 weeks, indicating that
the number of LPCs decreases during postnatal development.
Moreover, bipotential colonies derived from adult LPCs contained
substantially fewer albumin+ cells than those from neonatal LPCs. We
further examined the differentiation potential of neonatal and adult
LPCs by transplantation and found that neonatal cells differentiated
into mature hepatocytes in recipient livers more frequently than adult
LPCs. Since we previously reported that the transcription factor
grainyhead-like 2 (GRHL2) expressed in EpCAM+ cells inhibits
hepatocytic differentiation, we examined whether targets of GRHL2
might block hepatocytic differentiation. DNA and microRNA
microarrays revealed that miR122, the expression of which
correlates with hepatocytic differentiation, was greatly reduced in
adult as compared with neonatal EpCAM+ cells. Indeed, GRHL2
negatively regulates the promoter/enhancer activity of the Mir122
gene. Our results indicate that neonatal but not adult EpCAM+ LPCs
have great potential to produce albumin+ hepatocytes. GRHL2
suppresses transcription of miR122 and thereby restricts the
differentiation potential of adult LPCs.
KEY WORDS: Tissue stem/progenitor cells, Bipotential, Postnatal
development, Hepatocyte, Cholangiocyte, Transcription factor
INTRODUCTION
Many developing organs contain multipotent tissue-specific stem/
progenitor cells that may be extinguished later in development
(Zhou et al., 2007; Pan et al., 2013). Conversely, tissue-specific
stem/progenitor cells are thought to contribute to supplying cellular
components in mature organs for the maintenance of organ
homeostasis and for recovery from injury. In some adult tissues/
organs, such as the intestine and skin, it has been shown that
residential stem/progenitor cells continuously produce multiple cell
types in homeostasis (Fuchs, 2009; Barker et al., 2010). In many
epithelial tissues and organs, however, it is not clear whether tissueDepartment of Tissue Development and Regeneration, Research Institute for
Frontier Medicine, Sapporo Medical University School of Medicine, S-1, W-17,
Chuo-ku, Sapporo 060-8556, Japan.
*Author for correspondence ([email protected])
Received 31 May 2014; Accepted 10 September 2014
4448
specific stem/progenitor cells actually contribute to cellular turnover
throughout life.
The liver contains two types of epithelial cells, namely
hepatocytes and cholangiocytes (biliary epithelial cells), which
originate from fetal liver stem/progenitor cells (LPCs) termed
hepatoblasts (Oertel et al., 2003; Tanimizu et al., 2003). Under severe
chronic liver injury the ability of hepatocytes and/or cholangiocytes
to multiply is exhausted, and then adult LPCs may be activated to
supply these cell types. In addition to LPCs in regenerating livers, it
has been reported that adult LPCs exist in the normal liver. Adult
LPCs have been enriched in cellular fractions and express
cholangiocyte markers, including epithelial cell adhesion molecule
(EpCAM), CD133 (also known as PROM1), CD13 (also known as
ANPEP), SRY-related HMG box transcription factor 9 (SOX9) and
osteopontin (OPN; also known as SPP1) (Suzuki et al., 2008;
Kamiya et al., 2009; Okabe et al., 2009; Cardinale et al., 2011;
Dorrell et al., 2011; Furuyama et al., 2011; Español-Suñer et al.,
2012). By genetic lineage tracing it was demonstrated that SOX9+
LPCs forming the ductal plate, which constitutes the primitive
structure of the bile duct, actually supplied cholangiocytes as well as
mature hepatocytes (MHs) (Carpentier et al., 2011). By contrast,
recent results showed that adult LPCs barely supplied MHs in normal
liver (Malato et al., 2011; Español-Suñer et al., 2012). These results
suggest that LPCs may exist in or near bile ducts and that LPCs might
alter their differentiation potential during development.
In this study, we used EpCAM as a marker to enrich LPCs, which
form bipotential colonies in a clonal condition. We found that neonatal
EpCAM+ LPCs produce hepatocytes much more efficiently than adult
LPCs. We further demonstrated that grainyhead-like 2 (GRHL2), a
cholangiocyte-specific transcription factor, downregulates miR122,
a microRNA (miRNA) known to be important for hepatocytic
differentiation. Thus, our data indicate that one crucial transcription
factor is involved in defining the differentiation capability of epithelial
tissue-specific stem/progenitor cells.
RESULTS
Isolation of LPCs from late-fetal and neonatal livers
It has been reported that the number of hepatoblasts (fetal LPCs)
decreases from mid- to late fetal stage (Suzuki et al., 2002), that
adult LPCs exist in livers beyond 2 weeks after birth, and that their
number decreases during postnatal development (Kamiya et al.,
2009). In accordance with a previous report (Okabe et al., 2009),
we used EpCAM as a marker to enrich adult-type LPCs. We
immunohistochemically confirmed that EpCAM+ cells are localized
next to the portal vein after E17 (Fig. 1A). A single-cell suspension
was used to isolate single EpCAM+ cells from the CD45− TER119−
CD31− non-hematopoietic/non-endothelial cell fraction (Fig. 1B).
As shown in Fig. 1C, EpCAM+ cells formed bipotential colonies
containing albumin (ALB)+ hepatocytes and CK19+ cholangiocytes.
Interestingly, 1-week EpCAM+ cells formed bipotential colonies
most efficiently (Table 1 and Fig. 1D). By contrast, EpCAM+ cells
DEVELOPMENT
ABSTRACT
RESEARCH ARTICLE
Development (2014) 141, 4448-4456 doi:10.1242/dev.113654
Fig. 1. Colony formation by EpCAM+ cells at different
developmental stages. (A) Expression of EpCAM in E17,
1-week (1W) and 8-week (8W) livers. EpCAM expression
(green) is observed in primitive duct structures in E17 liver,
whereas it is expressed in bile ducts in 1W and 8W livers.
Scale bar: 50 μm. PV, portal vein. (B) Isolation of EpCAM+
cells. Single EpCAM+ cells were isolated from the
CD45− TER119− CD31− fraction, which contains nonhematopoietic/non-endothelial cells, by FACS. Density plots
for EpCAM+ cell isolation from neonatal livers are shown.
(C) Bipotential colonies derived from E17, 1W and 8W
EpCAM+ cells. Purified EpCAM+ cells were plated at a low
cell density. Typical bipotential colonies containing
both ALB+ CK19− hepatocytes (red) and ALB− CK19+
cholangiocytes (green) are shown. Bipotential colonies also
contain ALB+ CK19+ cells. Nuclei were counterstained with
Hoechst 33258. (D) Number of colonies derived from
EpCAM+ cells at different development stages. Bipotential
colonies were apparently formed from EpCAM+ cells by
4 weeks after birth. Thereafter, bipotential colonies were
formed only to a limited degree from EpCAM+ cells. Colonies
containing ALB+ CK19−, ALB+ CK19+, and ALB− CK19+ cells
were counted as bipotential colonies, whereas those only
containing ALB− CK19+ cells were counted as cholangiocyte
colonies. Top error bars indicate s.e.m. for the number of
bipotential colonies, whereas bottom error bars indicate
s.e.m. for cholangiocyte colonies.
Table 1. Colony formation from EpCAM+ cells isolated from developing
livers
Small (<50 cells)
Large (≥50 cells)
Age
Bipotential
Cholangiocytic
Bipotential
Cholangiocytic
E17
1W
2W
3W
4W
6W
8-11W
11.7±5.6
24.6±1.7
12.5±4.1
3.5±1.8
1.0±0.4
0.3±0.1
0.1±0.1
20.7±5.3
9.8±1.4
9.0±1.7
2.8±0.6
4.5±0.8
6.1±1.3
2.3±0.4
13.9±4.3
67.8±3.1
22.5±3.6
8.0±1.7
1.7±0.4
0.3±0.1
0.4±0.2
8.4±4.2
8.3±1.4
12.0±3.4
8.6±2.5
6.3±1.1
6.4±1.2
4.7±0.4
The numbers of colonies containing fewer than 50 cells (small) and more than
50 cells (large) per 5000 EpCAM+ cells are shown as the average±s.e.m. For
each experiment n=6-8 mice for E17-2W and n=3 mice for 3W-11W. The
colony assay was repeated at least three times. E, embryonic day; W, week.
compared with only ∼10% of cells in colonies derived from 6-week
EpCAM+ cells (adult).
Next, we analyzed the level of albumin expression in each
bipotential colony. For this purpose, we prepared cDNA from total
RNA extracted from each colony and examined whether each
colony expresses albumin (Fig. 2C). We then selected albumin+
colonies and quantified the albumin expression level. The level of
albumin expression was much higher in neonatal bipotential
colonies than in adult colonies (Fig. 2D). Expression of Ck19
(Krt19), Sox9 and Epcam was relatively low in colonies expressing
albumin at high level.
Neonatal LPCs differentiate to hepatocytes in vitro and
in vivo
To further clarify whether neonatal and adult LPCs can differentiate
into hepatocytes, we subcultured colonies derived from EpCAM+
cells isolated from 1-week and 6-week livers. We picked up ten
colonies of each and successfully established four and five clones
from 1-week and 6-week colonies, respectively. Two out of four
neonatal clones (clones 2 and 4) retained the original marker
EpCAM, whereas it was eventually lost from the other clones (data
not shown).
We induced hepatocytic differentiation of LPC clones by
oncostatin M (OSM) and Matrigel (MG). After induction, all
neonatal clones expressed marker genes related to differentiated
functions of hepatocytes, including metabolic enzymes and
cytochrome P-450s (Fig. 3A). By contrast, adult clones were
induced to express only albumin and carbamoyl-phosphate
synthetase 1 (Cps1). We quantified the expression level of Cps1
and tyrosine aminotransferase (Tat) and the data showed that
neonatal clones expressed both genes at a level comparable to
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DEVELOPMENT
isolated from 6- to 11-week livers mostly formed cholangiocyte
colonies containing only CK19+ cells (Fig. 1D). These results are
consistent with a previous report demonstrating that the number of
LPCs possessing the ability to form bipotential colonies decreases
during postnatal development (Kamiya et al., 2009). In addition, we
noted that the ratio of ALB+ to total colony-forming cells was much
lower in bipotential colonies derived from 8-week versus 1-week
EpCAM+ cells (Fig 1Cb,c). This suggests that, in addition to a
reduction in LPC numbers, the hepatocytic differentiation potential
of LPCs might decrease as postnatal development progresses.
To further compare the differentiation capability of neonatal and
adult LPCs, we examined the ratio of ALB+ cells, including both
ALB+ CK19− and ALB+ CK19+, among colony-forming cells
(Fig. 2A). The results indicated that more than 50% of cells were
ALB+ in colonies derived from 1-week EpCAM+ cells (neonate) as
RESEARCH ARTICLE
Development (2014) 141, 4448-4456 doi:10.1242/dev.113654
MHs (Fig. 3B). Neonatal cells showed hepatocytic morphology
after induction (Fig. 3Ca,b). Furthermore, the expression of
hepatocyte nuclear factor 4α (HNF4α), CCAAT/enhancer binding
protein α (C/EBPα), ALB and CPS1 was induced in neonatal
clones (Fig. 3Ca-h). Neither morphological changes nor the
expression of HNF4α and ALB was clearly induced in adult
clones (Fig. 3Ci-m). These results further support the proposal that
neonatal LPCs have greater potential than adult LPCs to
differentiate into hepatocytes.
In order to examine the differentiation capability of neonatal and
adult LPCs in vivo, we transplanted neonatal and adult clones. All the
clones were labeled with GFP, and GFP+ cells were enriched by FACS
(Fig. 4A). Recipient mice were pretreated with retrorsine, which
inhibits hepatocyte proliferation, and then 70% partial hepatectomy
was performed at the time of transplantation (Fig. 4B). Recipient liver
sections were stained with anti-GFP antibody to identify donor cells
(Fig. 4C). Engraftments of donor cells were observed in recipients
transplanted with either neonatal or adult clones (Table 2). Neonatal
clones were engrafted as hepatocytic or ductular cells (Fig. 4Ca,b).
By contrast, the engraftment of adult cells was rare and these were
mostly found as ductular cells (Fig. 4Cc).
We further examined the recipient livers transplanted with neonatal
cells by expression of hepatocyte and cholangiocyte markers
(Fig. 4D). Neonatal clones differentiated to HNF4α+ hepatocytes or
CK19+ cholangiocytes (Fig. 4Da-d). Since recipient CD31+ sinusoidal
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endothelial cells entered into a cluster of HNF4α+ donor cells, donorderived hepatocytes were incorporated into recipient liver trabeculae
(Fig. 4De-h). We also examined the expression of hepatocyte and
cholangiocyte markers in recipient livers transplanted with adult
cells and found that most of the donor cells differentiated to CK19+
ductular cells (Fig. 4Ea-d). The GFP+ ductular structure (dotted
circle in supplementary material Fig. S1a-c) was separate from the
portal veins (asterisks in supplementary material Fig. S1). The
differentiated cells also expressed other cholangiocyte markers
(supplementary material Fig. S1f-w). A small proportion of donor
cells became CK19+ HNF4α+ cells, which did not form the ductular
structure (Fig. 4Ee). These results indicate that EpCAM+ LPCs show
bidirectional differentiation capability in vivo, although adult LPCs
rarely differentiate into hepatocytes.
These results further support the proposal that the bipotency of
LPCs is altered during development. Thus, we considered that a
molecular machinery restricting the differentiation potential might
exist in adult LPCs.
Comparison between neonatal and adult EpCAM+ cells
It has been demonstrated that certain transcription factors are
crucial in defining cellular lineages. We previously found that
cholangiocyte transcription factors, including GRHL2 and SOX9,
are expressed at a higher level in adult than in neonatal EpCAM+
cells (Tanimizu et al., 2013). However, we had not examined
DEVELOPMENT
Fig. 2. Neonatal and adult LPCs differ in their potential
to produce albumin+ hepatocytes. (A) Bipotential
colonies contain two types of hepatocytic cells: ALB+
CK19− (solid arrowheads) and ALB+ CK19+ (open
arrowheads) cells. A bipotential colony derived from a
neonatal EpCAM+ cell is shown (a), with the boxed area
magnified to show individual and merged channels (b-d).
Scale bars: 50 μm. (B) Ratio of ALB+ cells among total
cells in each colony. More than 50% and ∼10% of cells
are ALB+ in each neonatal and adult colony, respectively.
Error bars indicate s.e.m. (C) Albumin expression in each
colony. Low cell density culture was performed for
2 weeks. Total RNA extracted from each colony was used
to synthesize cDNA to examine albumin expression by
PCR. Colonies positive for albumin were considered to
be derived from LPCs. Gapdh provides a control.
(D) Quantitative analysis of albumin, Ck19, Epcam and
Sox9 expression in each colony. Four albumin+ neonatal
colonies were selected, while all four albumin+ adult
colonies were used to quantify the expression level of
albumin and Ck19. Neonatal bipotential colonies express
more albumin than adult colonies. The expression levels of
Ck19 are similar in neonatal and adult colonies. Relative
expression levels compared with that of mature
hepatocytes (MHs), which were cultured for 1 day,
are shown.
RESEARCH ARTICLE
whether neonatal EpCAM+ cells equally expressed these markers at
low level or if they contain cells positive and negative for the
markers. We prepared smear samples of FACS-purified EpCAM+
cells and examined the expression of HNF1β, SOX9, GRHL2 and
HNF4α, as well as CK19 and OPN in each neonatal and adult
EpCAM+ cell (Table 3). Consistent with the data in Table 3,
HNF1β, SOX9 and OPN, but not HNF4α, are expressed in neonatal
and adult EpCAM+ cells in liver tissues (supplementary material
Figs S2 and S3). Interestingly, ∼25% of neonatal EpCAM+ cells
were GRHL2− (Fig. 5Aa-c), whereas the adult EpCAM+ fraction
barely contained any GRHL2− cells (Fig. 5Ad-f ).
We further confirmed that neonatal EpCAM+ cells were GRHL2+
(Fig. 5Bb-d) or GRHL2− (Fig. 5Be-g) by examining its expression
Development (2014) 141, 4448-4456 doi:10.1242/dev.113654
in neonatal liver sections. Neonatal EpCAM+ GRHL2− cells formed
smaller ductules in the periphery. By contrast, bile ducts in the adult
liver consist of EpCAM+ GRHL2+ cells (Fig. 5Bh-n). Additionally,
the expression pattern of CK19 was similar to that of GRHL2
(supplementary material Fig. S3). Previously, we demonstrated that
GRHL2 not only promotes the maturation of cholangiocytes but
also inhibits hepatocytic differentiation (Tanimizu et al., 2013).
Therefore, these results suggest that substantial differences in
differentiation potential might exist among neonatal EpCAM+ cells,
as well as between neonatal and adult cells. We further examined the
possibility that GRHL2 expression might regulate the hepatocytic
differentiation potential of LPCs.
Fig. 3. Neonatal EpCAM+ LPCs efficiently differentiate to functional
hepatocytes in vitro. (A) Expression of hepatocyte markers in cultures of
neonatal and adult clones. Neonatal and adult LPC clones were induced to
differentiate into hepatocytes by sequential treatment with oncostatin M (OSM)
and Matrigel (MG). Expression of marker genes was examined by PCR. In
neonatal cultures, metabolic enzymes and cytochrome P-450s were induced.
(B) Expression level of Cps1 and Tat in neonatal and adult LPCs after
hepatocytic differentiation. Both Cps1 and Tat were induced much more in
neonatal than in adult cells. The average values from four clones are shown.
Error bars represent s.e.m. (C) Hepatocytic differentiation of neonatal and adult
clones. Morphology of neonatal clone 4 shows round nuclei and dense
cytoplasm after inducing hepatocytic differentiation (a,b). Expression of the
hepatocyte markers HNF4α, C/EBPα, ALB and CPS1 is detectable (c-h). By
contrast, cells of adult clone 1 do not show hepatocytic morphology even after
inducing hepatocytic differentiation (i,j). Expression of HNF4α is weakly
observed. A cell positive for ALB is seen (arrowhead) but it is negative for
nuclear HNF4α (k-m). Scale bar: 50 μm.
To correlate the expression profile of GRHL2 with the bipotency of
LPCs, we examined its expression in colonies derived from neonatal
and adult EpCAM+ cells. We found that intense and uniform
expression of GRHL2 was maintained in colonies derived from
adult EpCAM+ cells (Fig. 6Aa-e). By contrast, neonatal EpCAM+
cells formed two types of colonies: one consisted of GRHL2−
cells (Fig. 6Af-j) and the other of GRHL2+ and GRHL2− cells
(Fig. 6Ak-o). Although the neonatal GRHL2− and GRHL2+
colonies were similar in size, the GRHL2− colonies contained
more ALB+ cells (Fig. 6B). In addition, in GRHL2+ colonies ALB
was restricted to cells in which GRHL2 was spontaneously
downregulated (Fig. 6C). Based on these results, we considered
the possibility that targets of GRHL2 might be involved in reducing
the hepatocytic differentiation potential of LPCs.
To identify molecules downstream of GRHL2, we performed
mRNA and miRNA microarray analyses using a hepatic progenitor
cell line HPPL with or without GRHL2. Since HPPL cells do not
express GRHL2, we expected that its targets would be clearly
upregulated or downregulated in HPPL cells with GRHL2. The
results of the mRNA microarray suggested that some metabolic genes,
such as arginase and Cps1 were downregulated by overexpression of
GRHL2. The results of the miRNA microarray, by contrast, suggested
that miR122 was remarkably downregulated by GRHL2 (Table 4;
supplementary material Table S3). We confirmed this result by
quantitative PCR analysis, showing that overexpression of GRHL2
substantially downregulated miR122 in HPPL cells (Fig. 7A).
It has been reported that miR122 is important for hepatocytic
differentiation (Laudadio et al., 2012). Consistently, overexpression
of GRHL2 in HPPL cells, in which miR122 was downregulated
(Fig. 7A), inhibited hepatocytic differentiation (supplementary material
Fig. S4). We also found that knockdown of GRHL2 increased
miR122 in adult EpCAM+ cells (Fig. 7B). We further examined the
effect of miR122 on hepatocytic differentiation by introducing a
miR122 mimic, and found that it slightly upregulated albumin
and downregulated Ck19 in colonies derived from adult EpCAM+
cells (Fig. 7C). We also found that the miR122 mimic upregulated
albumin, Cps1 and Tat but not Hnf4a in primary EpCAM+ cells
(supplementary material Fig. S5). Consistent with the notion that the
Notch signaling pathway promotes cholangiocyte differentiation
(Tanimizu and Miyajima, 2004; Zong et al., 2009), DAPT, which is
a γ-secretase inhibitor and thereby a potent inhibitor of the Notch
signaling pathway, further promoted upregulation of Cps1 and Tat
by miR122 (supplementary material Fig. S5). Consistent with
their reduced capacity to differentiate into hepatocytes, miR122
expression in adult EpCAM+ cells was significantly lower than in
neonatal cells, whereas Grhl2 expression in adult cells was higher
than in neonatal cells (Fig. 7D). Furthermore, in neonatal and adult
LPC colonies, Grhl2 and Mir122 expression levels are negatively
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DEVELOPMENT
GRHL2 suppresses the expression of miR122
RESEARCH ARTICLE
Development (2014) 141, 4448-4456 doi:10.1242/dev.113654
Fig. 4. Engraftment of neonatal and adult LPCs in recipient livers.
(A) Preparation of GFP+ LPCs. LPC clones were infected with
lentivirus containing the GFP gene. GFP+ cells were isolated by FACS
and expanded before transplantation. (B) Timecourse of retrorsine
treatments, partial hepatectomy and transplantation. (C) GFP+ cells
are detected in recipient livers. Neonatal clones are detected as
hepatocytic cells (solid arrowheads in a,b) as well as ductular cells
(open arrowheads in b). Adult donor cells are mostly observed as
ductular cells (c). Images represent recipient livers transplanted with
neonatal clone 2 (a), neonatal clone 4 (b) and adult clone 1 (c). GFP
expression was detected by DAB staining. (D) Neonatal LPC clones
differentiate into HNF4α+ hepatocytes and CK19+ cholangiocytes in
the recipient liver. Recipient liver sections were stained with anti-GFP,
anti-CK19 and anti-HNF4α (a-d) or with anti-GFP, anti-CD31 and antiHNF4α (e-h) antibodies. GFP+ donor cells differentiate into CK19+
cholangiocytes and HNF4α+ hepatocytes in the recipient liver (a-d).
GFP+ HNF4α+ hepatocytes are associated with recipient CD31+
endothelial cells, indicating that donor-derived hepatocytes are
incorporated into recipient liver tissue. Sections of a recipient liver
transplanted with neonatal clone 4 are shown. Nuclei were
counterstained with Hoechst 33258. (E) Adult LPC clone differentiates
to CK19+ cholangiocytes in the recipient liver. A frozen section of
recipient liver transplanted with adult clone 1 was stained with antiGFP, anti-CK19 and anti-HNF4α. GFP+ donor cells express CK19 and
form ductular structures. Some GFP+ CK19+ cells are positive for
HNF4α+ (arrowheads) and are not incorporated into ductular structure
(e, which is a magnification of the boxed region in d). Nuclei were
counterstained with Hoechst 33258. Scale bars: 100 μm in C; 50 μm
in D,E.
Table 2. Efficiency of cell transplantation
Donor cell spots/cm2
Clone
Donor cell
occurrence‡
With hepatocytic
features
With ductular
features
Neonate clone 2
Neonate clone 4
Adult clone 1
Adult clone 4
4/4
3/3
1/3
0/2
0.94±0.24*
1.4±0.3**
0.11±0.04
–
0.19±0.09*
1.4±0.1**
0.77±0.39
–
For each recipient liver, 8-12 sections were prepared and examined for GFP+
cells. Clusters containing more than two GFP+ cells were counted as donor cell
spots. Statistical analysis showed that neonatal clones were engrafted as
hepatocytes more efficiently than adult clones (neonatal clone 2 versus adult
clone 1, *P=0.026; neonatal clone 4 versus adult clone 1, **P=0.045).
‡
Number of mice with GFP+ donor cells/total number of mice transplanted.
4452
containing the Mir122 proximal promoter/enhancer (Fig. 7E). The
results indicate that GRHL2 negatively regulates Mir122 promoter/
enhancer activity.
DISCUSSION
Each tissue/organ may contain tissue-specific stem/progenitor cells
that contribute to organogenesis, homeostasis and the recovery from
injury. However, it remains unclear whether tissue stem/progenitor
cells retain the same differentiation potential throughout life. In this
study, by examining characteristics of LPCs continuously between
late fetal and adult periods, we demonstrate that the number of LPCs
and their differentiation capability are restricted during postnatal
liver development. We further identified that downregulation of
miR122 by GRHL2 is one of the molecular pathways regulating the
differentiation capability of LPCs.
We have shown (Fig. 7) that miR122 alone can increase the
expression of albumin in adult LPCs. However, given that
overexpression of GRHL2 remarkably inhibited hepatocytic
differentiation (supplementary material Fig. S4), the effect of
miR122 on hepatocytic differentiation is relatively weak. We
previously found that overexpression of GRHL2 inhibited the
expression of HNF4α in neonatal EpCAM+ cells, which key to
suppressing hepatocytic differentiation. However, introduction of a
miR122 mimic did not significantly increase Hnf4a expression in
DEVELOPMENT
and positively correlated with albumin expression, respectively
(supplementary material Fig. S6). These results further support
the hypothesis that downregulation of miR122 by GRHL2 is one
of the key molecular pathways restricting the hepatocytic
differentiation potential of LPCs.
It has been reported that Mir122 expression is controlled by the
proximal promoter and enhancers (Xu et al., 2010; Laudadio et al.,
2012). In order to determine whether GRHL2 transcriptionally
regulates Mir122, we examined its effect on reporter constructs
RESEARCH ARTICLE
Development (2014) 141, 4448-4456 doi:10.1242/dev.113654
Table 3. Expression of cholangiocyte and hepatocyte markers in neonatal and adult EpCAM+ cells
CK19
Neonate
%
n
Adult
%
n
GRHL2
HNF1β
OPN
SOX9
ALB
HNF4α
–
+
–
+
–
+
–
+
–
+
–
+
–
+
4.4
250
95.6
26
373
74
1.3
78
98.7
1.0
102
99
3.0
562
97.0
97.7
526
2.3
0.8
634
99.2
0
155
100
1.8
227
98.2
0.9
218
99.1
0
234
100
1.8
227
98.2
99.3
424
0.7
100
112
0
adult EpCAM+ cells (supplementary material Fig. S5). Taken
together, we consider that, in addition to miR122, other downstream
targets of GRHL2 also participate in modulating the differentiation
potential of LPCs.
LPCs have been thoroughly characterized beyond 2 weeks after
birth (Kamiya et al., 2009). In addition, by examining the
differentiation potential of late fetal, neonatal and adult LPCs, we
Fig. 5. Expression of GRHL2 in adult and neonatal EpCAM+ cells. (A) The
neonatal EpCAM+ fraction contains GRHL2+ and GRHL2− cells. Some
neonatal EpCAM+ cells are GRHL2− (arrowhead in a-c), whereas adult
EpCAM+ cells do not contain GRHL2− cells (d-f ). EpCAM+ cells were isolated
by FACS and used to prepare smear samples. (B) EpCAM+ GRHL2− and
EpCAM+ GRHL2+ cells form bile ducts in neonatal livers. In neonatal liver, bile
ducts associated with a clear lumen express GRHL2 (b-d), whereas it is not
expressed by some of the small ductules (e-g). By contrast, GRHL2 is
expressed in small ductules (l-n) as well as large ducts (i-k) in adult liver. Boxed
regions in a and h are enlarged in b-g and i-n, respectively. Scale bars: 50 μm
in A and Bg,n; 100 μm in Ba,h.
found that neonatal LPCs showed a strong ability to differentiate
into functional hepatocytes in vitro and in vivo, suggesting that
EpCAM+ LPCs are a candidate for supplying hepatocytes during
early postnatal development as embryonic ductal plate cells
(Carpentier et al., 2011). However, since hepatocytes show the
ability to proliferate even in a clonal condition by 4 weeks (data not
shown), they certainly play an important role in supplying new
hepatocytes during early postnatal development. Therefore, further
experiments are necessary to prove whether neonatal EpCAM+
LPCs also supply new hepatocytes during development.
Adult LPCs have been enriched in cellular fractions that are
positive for cholangiocyte markers and they have potential to
differentiate to hepatocytes in vitro and in vivo (Suzuki et al., 2008;
Okabe et al., 2009; Cardinale et al., 2011; Dorrell et al., 2011;
Furuyama et al., 2011; Español-Suñer et al., 2012). Consistent with
previous work, we demonstrated that adult LPCs are enriched in the
EpCAM+ fraction. However, compared with 1-week LPCs, their
capability to differentiate into hepatocytes was very low. Given that
recent lineage-tracing studies failed to detect MHs derived from
LPCs in vivo without injury (Español-Suñer et al., 2012), the
differentiation capability of LPCs appears extremely limited at the
late postnatal and adult period. Specific stimulation, including Wnt
plus R-spondin (Huch et al., 2013), and/or inhibition of the Notch
signal (Boulter et al., 2012) might be necessary to release adult
LPCs from the molecular machineries that serve to block
hepatocytic differentiation.
By examining the expression of marker genes at the single-cell
level, we found that neonatal EpCAM+ cells can be divided into
GRHL2− and GRHL2+. Interestingly, EpCAM+ GRHL2− cells
formed small ductules in the periphery of the neonatal liver.
According to the recent model of bile duct development, in which
development/morphogenesis proceed from the liver hilum to the
periphery (Antoniou et al., 2009), these EpCAM+ GRHL2− cells
might be recently committed from hepatoblasts and thereby retain
bidirectional differentiation capability. In addition, in contrast to
adult cells, neonatal EpCAM+ GRHL2+ cells spontaneously lose
expression of GRHL2 and then express ALB during clonal
expansion (Fig. 6C). Therefore, it can be assumed that GRHL2
expression is not necessarily fixed even in EpCAM+ GRHL2+ cells
at neonatal stage, and, thereby, that neonatal EpCAM+ GRHL2+
cells retain the potential to differentiate into hepatocytes.
In this study, we demonstrated that neonatal and adult LPCs are
distinct in terms of their hepatocytic differentiation potential. It has
been demonstrated that the combination of GATA4, FOXA3 and
HNF1α (Huang et al., 2011) or of HNF4α and one of the FOXA
factors (Sekiya and Suzuki, 2011) converts fibroblasts into
hepatocytes. By contrast, overexpression of HNF4α in the presence
of endogenous FOXA1 did not induce hepatocytic characteristics in
EpCAM+ cells (Tanimizu et al., 2013). Given that recent reports
4453
DEVELOPMENT
EpCAM+ cells were isolated from neonatal and adult livers by FACS, smear samples were prepared and used for immunostaining. The indicated number of
EpCAM+ cells were examined for expression of each marker. Ratios of marker-negative and marker-positive cells are shown.
RESEARCH ARTICLE
Development (2014) 141, 4448-4456 doi:10.1242/dev.113654
Fig. 6. Change in GRHL2 expression during clonal
expansion of EpCAM+ cells. (A) Adult LPCs maintain
GRHL2 during clonal culture. GRHL2 is expressed in
adult colonies at day 6 and 14 (a-e). By contrast,
neonatal colonies are either GRHL2+ (f-j) or GRHL2−
(k-o). Boxed regions in c, h, m and k are enlarged in
d,e, i,j, n,o and k′,l′, respectively. Scale bars: 500 μm in
c,h,m; 100 μm in b,l. (B) GRHL2 expression does not
affect the clonal proliferation of LPCs but their
differentiation potential. Neonatal colonies were stained
with anti-albumin, anti-GRHL2 antibody and Hoechst
33258. Total and albumin+ cells in each colony were
counted. Error bars represent s.e.m. (C) Expression of
GRHL2 and ALB are mutually exclusive in neonatal
GRHL2+ colonies. The boxed region in c is magnified in
d-f. Scale bar: 50 μm.
suggested that, even in chronically injured livers, LPCs do not
efficiently supply new hepatocytes (Tarlow et al., 2014), adult LPCs
might be equipped with molecular machineries blocking hepatocytic
differentiation. The present finding that miR122 is downregulated by
GRHL2 might provide an explanation for such mechanisms. Recently,
it was demonstrated that key transcription factors defining neural
lineage cells, including PAX6 and ETV6, inhibit dedifferentiation
induced by OCT3/4 (POU5F1), SOX2, KLF4 and cMYC during
reprograming (Hikichi et al., 2013). As we have previously
demonstrated (Senga et al., 2012; Tanimizu et al., 2013), GRHL2 is
a key transcription factor in inducing the cellular properties of mature
cholangiocytes. Therefore, transcription factors conferring specific
functions and/or structures to certain cell types might generally define
the differentiation potential of tissue-specific stem/progenitor cells. In
order to use highly proliferative LPCs in vitro and in vivo for the
purposes of producing functional hepatocytes, it might be important to
release them from inhibitory machineries in order to make them
responsive to inductive signals for hepatocytic differentiation.
Cell isolation and sorting
E17 mouse livers were cut into small pieces and then digested in PBS
containing Mg2+, Ca2+ and Librase-TM (Roche Applied Sciences). For
neonatal and adult mouse livers, a two-step collagenase perfusion method
was used to isolate cells as previously reported (Tanimizu et al., 2013).
Liberated cells were used to isolate EpCAM+ cells by FACS. Non-specific
binding of antibodies was blocked by antibody against Fcγ receptor
(anti-CD16/CD32 antibody). Cells were labeled with FITC-conjugated antiEpCAM, APC-conjugated anti-CD45 (PTPRC), APC-conjugated antiCD31 (PECAM1) and APC-Cy7-conjugated anti-TER119 (LY76)
antibodies, and with propidium iodide (PI; Sigma-Aldrich). EpCAM+
cells were isolated from PI− live cells by FACS using a FACSAria II (BD
Biosciences). Antibodies used are listed in supplementary material
Table S1.
Extracellular matrix, growth factors and chemicals
Growth factor-reduced Matrigel (MG) and purified laminin 111 were from
BD Biosciences. Epidermal growth factor (EGF), hepatocyte growth factor
(HGF) and oncostatin M (OSM) were purchased from R&D Systems.
Table 4. miRNAs downregulated by GRHL2 overexpression
miRNA
GRHL2 versus control
miR122
miR760-5P
miR192
miR194
miR181B
0.078
0.29
0.35
0.38
0.38
A liver progenitor cell line, HPPL, was introduced with Grhl2 and the
expression profile of miRNAs compared between HPPL (control) and HPPLGrhl2 by microarray.
Single EpCAM+ cells were isolated by FACSAria II as described above.
Five to eight thousand cells were plated in a 35-mm dish coated with
laminin 111 (BD Biosciences). The cells were cultured in DMEM/Fischer
F-12 medium supplemented with 10% FBS, 10 ng/ml EGF, 10 ng/ml HGF,
0.1 μM dexamethasone (Dex; Sigma-Aldrich) and 1× insulin-transferrinselenium (ITS; Gibco). After 9 days of culture, cells were fixed in 4%
paraformaldehyde (PFA) and stained with anti-cytokeratin 19 (CK19)
and anti-albumin antibodies (supplementary material Table S1). Signals
were visualized with Alexa Fluor-conjugated secondary antibodies
(Molecular Probes). Nuclei were counterstained with Hoechst 33258
(Dojindo). Images were acquired on a Nikon X-81 fluorescence
microscope. Colonies containing more than 50 cells were categorized
into three groups: bipotential colonies, which consist of ALB+ hepatocytes
and CK19+ cholangiocytes; hepatocyte colonies, which consist of only
ALB+ cells; and cholangiocyte colonies, which consist of only CK19+
cells. We define a cell that forms bipotential colonies as an LPC in the
current study.
DEVELOPMENT
Colony assay
MATERIALS AND METHODS
4454
C57BL6 and nude mice were purchased from Sankyo Labo Service
Corporation (Tokyo, Japan). All animal experiments were approved by the
Sapporo Medical University Institutional Animal Care and Use Committee
and were carried out under the institutional guidelines for ethical animal use.
RESEARCH ARTICLE
Development (2014) 141, 4448-4456 doi:10.1242/dev.113654
Fig. 7. GRHL2 downregulates miR122. (A) Quantitative analysis of
miR122 expression in HPPL cells with or without GRHL2.
(B) Quantitative analysis of miR122 expression in adult EpCAM+ cells
with or without RNAi against Grhl2. (C) miR122 upregulates and
downregulates albumin and Ck19, respectively, in adult LPC colonies.
Adult EpCAM+ cells were cultured at low density for 2 weeks to allow
them to form colonies. Each colony was picked and split into two wells
of a 96-well plate for introduction of control or miR122 mimic. Two
days later, expression of albumin and Ck19 was examined by
quantitative PCR. Relative expression levels against MHs cultured for
1 day were calculated. The average values from three colonies are
shown. (D) Expression of miR122 during cholangiocyte
differentiation. miR122 is significantly downregulated in EpCAM+ cells
during postnatal development. (E) Negative regulation of Mir122 by
GRHL2. Mir122 promoter and enhancer sequences were linked to
firefly luciferase and HEK293T cells were used to examine their
activity in the absence and presence of GRHL2. GRHL2 was found to
significantly downregulate the promoter/enhancer activity of Mir122.
Error bars indicate s.e.m.
Induction of hepatocyte differentiation
To induce neonatal and adult LPC clones to differentiate to hepatocytes,
50,000 cells were plated on gelatin-coated wells of 24-well plates. After
cells became confluent, they were incubated with 10 ng/ml OSM, 1%
DMSO, 0.1 μM Dex and 1× ITS for 4 days and then overlaid with 5%
MG for an additional 4 days. Stealth RNA against Grhl2 (Invitrogen)
and miR122 mimic (Ambion) were introduced into cells using
RNAiMAX (Invitrogen).
PCR
Total RNA was isolated from purified EpCAM+ cells using an RNeasy Mini
Kit (Qiagen). cDNA was synthesized using the Omniscript Reverse
Transcription Kit (Qiagen). Primers used for PCR are listed in
supplementary material Table S2. TaqMan probes for Hprt, Cps1, Grhl2,
miR122, Sox9, Tat and U6 snRNA were purchased from Applied Biosystems
and used for quantitative PCR.
Cell transplantation
Nude mice were used for cell transplantation as described previously
(Kamiya et al., 2009). LPC clone cells were labeled by GFP-encoding
lentivirus vector and GFP+ cells were isolated by FACSAria II. Recipient
4-week nude mice were intraperitoneally injected with 60 mg/kg retrorsine
(Sigma-Aldrich) four times each week and then 70% of the liver was
surgically removed at transplantation. GFP+ cells (5×105-1×106) were
injected into spleen. Six weeks after the transplantation, the liver was
collected to examine the engraftment of donor cells. For detecting GFP+
cells in recipient livers, frozen sections were incubated with anti-GFP
antibody and then GFP signals were visualized using 3,3′-diaminobenzidine
(DAB) (Sigma-Aldrich) as a chromogenic substrate for horseradish
peroxidase, which was conjugated to anti-rabbit IgG antibody. Areas of
sections were measured using Olympus CellSense Software.
Immunofluorescence
Liver tissues were fixed in PBS containing 4% PFA or in Zamboni solution
at 4°C overnight. After washing in PBS and in PBS containing 20% sucrose,
they were embedded in OCT compound (Sakura Finetek). Thin sections
were prepared with a cryostat (CM1950, Leica Microsystems). After
permeabilization with 0.2% Triton X-100 and blocking with Blockace (DS
Pharma), sections were incubated with primary antibodies (supplementary
material Table S1). Images were obtained with Zeiss 510 and 780 confocal
microscopes.
Vector construction and luciferase assay
Grhl2 cDNA was amplified and inserted into a pcDNA3-myc vector, which
was generated by inserting a myc-tag into pcDNA3 (Life Technologies).
Mir122 proximal promoter and enhancer sequences were amplified using
mouse genomic DNA as a template and inserted into pUC118 vector
(Takara Bio). The promoter and enhancer were inserted into the KpnI/XhoI
4455
DEVELOPMENT
For establishing clones, each colony raised in the low density culture was
treated with trypsin after being surrounded by a cloning ring (Asahi Glass
Co., Tokyo, Japan). Cells were then plated in a well of a 24-well plate coated
with laminin 111 and kept in the medium that was used for the colony assay.
Expanded cells were replated every 4 days.
sites of a pGL4.10 vector (Promega), which contained minimal TK
promoter. HEK293T cells were co-transfected using pcDNA3 with or
without Grhl2, pGL4.10 with or without Mir122 promoter/enhancer, and a
pRL-TK vector containing Renilla luciferase. At 48 h after transfection,
luciferase activity was measured with the Dual-Luciferase Reporter Assay
System (Promega) on an Infiniti M1000-Pro microplate reader (Tecan).
Statistical analysis
Statistical analyses to determine s.e.m. for colony assay, qPCR and
luciferase assay and two-tailed Student’s t-tests were performed using Excel
(Microsoft).
Microarray analysis
Total RNA including microRNAs was extracted from HPPL cells infected
with pMXs-Neo or with pMXs-Neo-Grhl2 using miRNeasy (Qiagen).
Hybridization and data analysis were performed by Miltenyi Biotec using
miRXplore. Microarray data are available at GEO under accession number
GSE62862.
Acknowledgements
We thank Ms Minako Kuwano and Ms Yumiko Tsukamoto for technical assistance.
Competing interests
The authors declare no competing financial interests.
Author contributions
N.T.: conception and design, financial support, the collection, assembly, analysis
and interpretation of data, manuscript writing. S.K.: collection and assembly of data.
N.I.: discussion of data. T.M.: financial support, data interpretation, manuscript
editing.
Funding
This work is supported by the Ministry of Education, Culture, Sports, Science and
Technology, Japan, Grants-in-Aid for Young Scientists (B) for N.T. [22790386] and
Grants-in-Aid for Scientific Research (B) for T.M. [21390365, 24390304].
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.113654/-/DC1
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